Transcript Lab Report: GARP 2 & Stains-All studies Fernanda Balem Department of Pharmacology

Lab Report:
GARP 2 & Stains-All studies
Fernanda Balem
Department of Pharmacology
12/19/05
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Aim
 To investigate if Stains all dye could be used
to explore the conformations of GARP-protein.
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Stains all

Metachromatic cationic carbocyanine dye “Stains-all” (1ethyl-2-{3-(1-ethyl-naphthol[1,2-d]thiazoline-2-ylidine)-2methylpropenyl}
 It can bind to highly acidic proteins .

It can also be used to distinguish calcium-binding proteins
(CaBP) from others. CaBP are stained blue or purple by Stainsall while others proteins are stained red or pink
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Spectrum in ethylene glycol
Stains all with Ethylene Glycol
1
Absorbance
0.8
β
3%
0.6
10%
30%
α
0.4
60%
98%
0.2
7.9M in Eth.Glycol
673
642
612
581
550
520
489
459
428
400
0
-0.2
Wavelength
All the further experiments were conducted in
30% ethylene glycol .
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Interaction of Calmodulin with
Stains-all
5
673
642
612
581
550
520
489
459
428
0
6
673
642
612
4
5
6
7
Wavelength
581
4
3
550
3
520
0.5
2
489
2
1
459
Absorbance
1
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
400
Base Line
400
Absorbance
1.5
-0.5
Calmodulin3x diluted - difference spectra
B
428
Calmodulin 3x Diluted
A
Wavelength
Calmodulin Peaks
C
1
634nm
0.5
570nm
0
-0.5
50
25
12.5
6.25
3.12
1
530nm
490nm
-1
A- visible spectrum of Calmodulin with Stains all complexes with 2mM MOPS,30% ethylene glycol,pH 7.2. The dyeCalmodulin mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively. B- Shows the difference spectra
from Stains all/Calmodulin. C- Prominent peaks of difference spectra.
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Interaction of Calmodulin +
CaCl2 with Stains-all
0.6
673
642
612
581
550
520
489
459
428
5
6
673
642
6
-0.8
Wavelength
0.5
648nm
576nm
0
50
-1
612
5
-0.6
Calm +CaCl2 Peaks
C
-0.5
4
-0.4
7
Wavelength
581
3
-0.2
550
4
2
0
520
3
1
0.2
489
2
0.4
459
Absorbance
1
400
BL
400
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
Calm3x diluted+CaCl2-difference spectra
B
428
Calmodulin 3x diluted + CaCl2
A
25
12.5
6.25
3.12
1
554nm
533nm
493nm
A- visible spectrum of Calmodulin + CaCl2 with Stains all complexes with 2mM MOPS,30% ethylene glycol, pH 7.2.
The dye-CAlmodulin mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively. B- Shows the difference
spectra from Stains all/Calmodulin + CaCl2. C- Prominent peaks of difference spectra.
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Spectra of Stains-all in different concentrations
of Ethylene Glycol
The spectra was checked with 200mM Mops. 1:5%; 2:10%; 3:20%; 4:30%; 5:40%; 6:50%; 7:60%;
8:70%; 9:80%; 10:90% and 11:100%.
Dye concentration is 5μM.
Dye concentration is 15μM.
Dye concentration is 10μM.
Dye concentration is 20μM
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Dye/protein ratio: 1:50; 2:25; 3:12.5; 4:6.25; 5:3.12; 6:1.56; 7:1; 8:0.
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Spectra of Stains-all + Calmodulin
Fig A: Shows the spectra of dye/protein ratio of 12.5. This was
performed with fresh Stains-all.
Fig B: Shows the spectra of dye/protein ratio 12.5. Stains-all used was
1 week old. Dye concentration is 20μM and Protein 1.6μM.
B
1.2
2.5
1
Wavelength
673
642
612
673
642
612
581
0
489
0
459
0.2
428
0.5
581
0.4
550
1
0.6
520
3x diluted
489
1.5
0.8
459
Original
428
2
Absorbance
3
12.5 dye/calm
400
Stains all + Calm.
400
Absorbance
A
550

520

Wavelength
9
Time course experiment to find the minimum time required for
interaction of protein with stains all, we looked at increase in
absorbance at 650nm , as this band is induced by the resulting
interaction.
Spectra of SA/Calmodulin (cicle every 10 min)
650nm - 700nm
0
10
1.6
20
30
50
1
60
70
0.8
80
100
0.6
110
0.4
120
0.2
150
0
170
Wavelength
673
642
612
581
550
520
489
459
428
160
180
190
Absorbance
40
1.2
400
Absorbance
1.4
0.25
0.23
0.21
0.19
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0.03
0.01
-0.01
0 10 20 30 40 50 60 70 80 90 10 11 12 13 14 15 16 17 18 19
0 0 0 0 0 0 0 0 0 0
time in minutes
10


From our previous experiments, we conclude that dye/protein ratio of 12.5 is optimal
to induce the 650nm band.In order to make this experiment work for less
concentration of protein, different concentration of stains all was tried keeping the
ration of dye/protein 12.5
The experiment showed that 20μM dye is the minimum concentration, where we can
induce the 650nm band. For that we decided carry out all further experiments with a
dye concentration of 20μM.
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The experiments stopped working as the stains-all
might have got illuminated. So, We started the
experiment with new stains all and the results looks
different.
SA/Calmodulin - different ratio
1.4
1.2
1
0.8
0.6
0.4
0.2
673
642
612
581
550
520
489
459
428
0
-0.2
400

Ratio dye/protein: 2:50%; 3:25%; 4:12.5%; 5:6.25%; 6:3.12%;
7:1.15% and 8:1%.
The 650nm band is induced at 1.15 dye/protein ratio instead of 12.5
observed in previous experiments.
Absorbance

1
2
3
4
5
6
7
8
Wavelength
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Future plans
•We plan to investigate about the interaction of Garp-2
with stains all dye to help us understand if this dye could
be used as a system to find different conformation of the
Garp-2 protein.
•We are trying to find optimum buffer conditions to
concentrate Garp-2 for NMR studies.
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Acknowledgements
Dr. Judith
 Harpreet
 David

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Thank you very much for
your attention!
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